Localized to axonal initial segments, nodes of Ranvier, and neuromuscular junctions, voltage-gated sodium channels (VGSC) are almost exclusively responsible for the rising phase of action potentials. Either one or two (3 subunits can interact with a single pore-forming a-subunit to form the active VGSC on the cell surface. p subunits of the VGSC regulate both sodium channel density and function in adult neurons. We have recently identified the (32-subunit (02) of the VGSC as a substrate for a-, P-, and y-secretases involved in processing of the Alzheimer's disease (AD)-associated amyloid precursor protein. Mutations in both a- and p-subunits of the VGSCs have been linked to epileptic symptoms, induced by both decreased and increased VGSC activity resulting in an imbalance in sodium channel function. Interestingly, increased incidence of epileptic seizures has been associated with Alzheimer's disease. Our preliminary data indicate that increased BACE1- and PS1/y-secretase-mediated processing of 02 regulates VGSC a-subunit NaJ.1 mRNA and protein levels in neuronal cells, BACE1 transgenic mice, and in AD patients with elevated levels of BACE1 in brain. Elevated BACE1 activity inhibits VGSC function in neuronal cells and adult mouse hippocampal neurons. Here we propose to address the hypothesis that BACE1 and PS/v-secretase regulate VGSC function by release of P2-ICD and transcriptional regulation of a-subunit(s). Specific Aim 1 will focus on the biological function of p-/v-secretase-mediated processing of p2. We will determine the exact BACE1 and PS/y-secretase cleavage sites in P2 using in vitro and cell-free cleavage assays followed by MALDI-TOF MS analysis. We will also distinguish between potential y- and e-cleavages in (32, mediated by PS/y-secretase. Cytoplasmic and nuclear binding partners of P2-ICD will be identified by immunoprecipitation and P2-ICD affinity chromatography. To characterize effects of p-/y-secretase-mediated processing of P2 on gene expression, gene expression profiles will be obtained from full-length P2, 32-CTF, and |32-ICD-transfected neuronal cell lines. P2- ICD-DNA association will also be investigated. In Specific Aim 2, we will determine how p-/y-secretase-mediated processing of (32 specifically regulates VGSC a-subunit levels. Nav1.1 promoter sequences, which mediate P2 transactivation, will be identified using luciferase reporter assays and sequential Nav1.1 promoter deletions. We will determine the subcellular compartment where VGSC a-subunits accumulate following increased p-/y- secretase-mediated processing of P2 in neuronal cells and in primary neurons. P2"'" mice and BACE1 transgenic mice will be crossed to test for in vivo confirmation that P2 misprocessing induces VGSC a-channel Nav1.1 expression. VGSC a-subunit expression and adult hippocampal neuron sodium channel activity will be assessed in brains from BACEHfiZ^ animals. Since BACE1 activity and levels are significantly increased in AD brains, consequent dysfunction in VGSC activity may contribute to AD pathogenesis. Moreover, our preliminary data suggest that BACE1 and/or y-secretase inhibitors could affect VGSC function, and be beneficial in normalizing membrane excitability in AD patients with elevated BACE1 activity.